Hans F, Fiesel FC, Strong JC, Jaeckel S, Rasse TM, Geisler S, Springer W, Schulz JB, Voigt A, Kahle PJ

TDP-43 characterizes insoluble protein aggregates in distinct subtypes of frontotemporal lobar degeneration (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). TDP-43 mediates many RNA processing steps within distinct protein complexes. Here we identify novel TDP-43 protein interactors in a yeast 2-hybrid screen using an adult human brain cDNA library. We confirmed the ...

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TDP-43 interaction of seven hits by co-immunoprecipitation and assessed their co-localization in HEK293E cells. As pathological TDP-43 is ubiquitinylated, we focused on the ubiquitin-conjugating enzyme UBE2E3 and the ubiquitin isopeptidase UBPY. When cells were treated with proteasome inhibitor, ubiquitinylated and insoluble TDP-43 species accumulated. All three UBE2E family members could enhance the ubiquitinylation of TDP-43, whereas catalytically inactive [C145S]UBE2E3 was much less efficient. Conversely, silencing of UBE2E3 reduced TDP-43 ubiquitinylation. We examined 15 of the 34 known pathogenic TDP-43 mutants and found that one was excessively ubiquitinylated. This strong [K263E]TDP-43 ubiquitinylation was further enhanced by proteasomal inhibition as well as UBE2E3 expression. Conversely, UBE2E3 silencing and expression of UBPY reduced [K263E]TDP-43 ubiquitinylation. Moreover, wild-type but not active site mutant UBPY reduced ubiquitinylation of TDP-43 C-terminal fragments and of a nuclear import impaired mutant. In Drosophila melanogaster, UBPY silencing enhanced neurodegenerative TDP-43 phenotypes and the accumulation of insoluble high molecular weight TDP-43 and ubiquitin species. Thus, UBE2E3 and UBPY participate in the regulation of TDP-43 ubiquitinylation, solubility, and neurodegeneration.

Date: May. 13, 2014

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